Naturally occurring mutations in humans as well as genetic mouse models clearly indicate the necessity of pituitary follicle-stimulating hormone (FSH) in female reproductive cyclicity and fertility. Disruption or deletion of the FSH receptor gene or the beta subunit of the FSH (FSH beta) protein arrest ovarian follicle maturation at the pre-antral stage, thereby preventing ovulation. The activins and inhibins are potent regulators of pituitary FSH. The activin B isoform is produced within pituitary gonadotropes and acts in an autocrine or paracrine manner to stimulate FSHbeta subunit gene transcription. The mechanisms through which the activins mediate this effect are not understood. In other cell systems, activins bind to a heteromeric receptor complex consisting of one of the two activin type II receptors and the activin type IB receptor (ALK4). Upon ligand binding, the type II receptor phosphorylates ALK4, which phosphorylates the intracellular signaling molecules, SMAD2 and SMAD3. The mouse gonadotrope cell line, LbetaT2, produces FSHbeta mRNA and secretes FSH in response to activins. Over-expression of SMAD3, but not SMAD2, similarly stimulates FSHbeta gene expression in this cell line. These data suggest that activins may signal through SMAD3 to stimulate the FSHbeta gene. The first project in this proposal is designed to test this hypothesis by blocking SMAD3-dependent processes. First, SMAD2 or SMAD3 protein production will be inhibited in LbetaT2 cells using RNA interference (RNAi). It is predicted that the attenuation of SMAD3, but not SMAD2, production in these cells will block activin-stimulated FSHbeta expression. Second, cells will be transfected with dominant-negative (dn) forms of SMAD2 or SMAD3 that cannot be activated by ALK4 and thereby disrupt downstream signaling, dn-SMAD3, but not dn-SMAD2, is predicted to disrupt activinstimulated FSHbeta gene expression. Once in the nucleus, activated SMADs affect gene transcription through interaction with cell-specific DNA binding partners. It is hypothesized that SMAD3 interacts with gonadotrope-restricted transcription factors to stimulate FSHbeta subunit gene expression. The second project in this proposal is designed to test this hypothesis by using a yeast two-hybrid screen to identify SMAD3-interacting proteins in gonadotrope cells. Candidate proteins will then be investigated for their gonadotrope specific expression and their ability to interact with SMAD3 in mammalian cells. The results of the proposed studies will increase our understanding of mechanisms of FSH regulation by activins, may highlight novel targets for contraceptive design, and may provide insights into causes of some forms of infertility.